The present invention relates to a compound comprised of a hydrophilic polymer, such as polyethyleneglycol, cleavably linked to an amine-containing ligand, which in preferred embodiments can be an amine-containing lipid, drug or protein. The compounds are cleavable under mild thiolytic conditions to regenerate the amine-containing ligand in its original form.
Hydrophilic polymers, such as polyethylene glycol (PEG), have been used for modification of various substrates, such as polypeptides, drugs and liposomes, in order to reduce immunogenicity of the substrate and/or to improve its blood circulation lifetime.
For example, parenterally administered proteins can be immunogenic and may have a short pharmacological half-life. Proteins can also be relatively water insoluble. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients. Conjugation of PEG to proteins has been described as an approach to overcoming these difficulties. Davis et al. in U.S. Pat. No. 4,179,337 disclose conjugating PEG to proteins such as enzymes and insulin to form PEG-protein conjugates having less immunogenicity yet which retain a substantial proportion of physiological activity.
Veronese et al. (Applied Biochem. and Biotech, 11:141-152 (1985)) disclose activating polyethylene glycols with phenyl chloroformates to modify a ribonuclease and a superoxide dimutase. Katre et al. in U.S. Pat. Nos. 4,766,106 and 4,917,888 disclose solubilizing proteins by polymer conjugation. PEG and other polymers are conjugated to recombinant proteins to reduce immunogenicity and increase half-life. (Nitecki et al., U.S. Pat. No. 4,902,502; Enzon, Inc., PCT/US90/02133). Garman (U.S. Pat. No. 4,935,465) describes proteins modified with a water soluble polymer joined to the protein through a reversible linking group.
However, PEG-protein conjugates described to date suffer from several disadvantages. For example, modification of the protein with PEG often inactivates the protein so that the resulting conjugate has poor biological activity. Typically in the prior art to date, it is desired to have the PEG stably linked to the protein so that the beneficial properties provided by PEG remain. Another problem with some protein PEG conjugates is that upon decomposition of the conjugate undesirable products may be formed.
PEG has also been described for use in improving the blood circulation lifetime of liposomes (U.S. Pat. No. 5,103,556). Here, the PEG is covalently attached to the polar head group of a lipid in order to mask or shield the liposomes from being recognized and removed by the reticuloendothelial system. Liposomes having releasable PEG chains have also been described, where the PEG chain is released from the liposome upon exposure to a suitable stimulus, such as a change in pH (PCT/US97/18813). However, release of the PEG chain from the products are chemically modified and can have unpredictable, potentially negative effects in vivo.
Accordingly, it is an object of the invention to provide a compound where a ligand is covalently yet reversibly linked to a hydrophilic polymer. Upon cleavage of the linkage, the ligand in its native form is regenerated.
In one aspect, the invention includes a compound having the general structure: 
wherein R1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety; R2 is selected from the group consisting of H, alkyl and aryl; R3 is selected from the group consisting of O(Cxe2x95x90O)R4, S(Cxe2x95x90O)R4, and O(Cxe2x95x90S)R4; R4 comprises an amine-containing ligand; and R is selected from the group consisting of H, alkyl and aryl; and where orientation of CH2xe2x80x94R3 is selected from the ortho position and the para position.
In one embodiment, R5 is H and R2 is selected from the group consisting of CH3, C2H5 and C3H8. In another embodiment, R2 and R5 are alkyls.
In another embodiment, the amine-containing ligand R4 is selected from the group consisting of a polypeptide, an amine-containing drug and an amine-containing lipid. In an embodiment where the amine-containing ligand R4 is an amine-containing lipid, the lipid includes either a single hydrocarbon tail or a double hydrocarbon tail. In one preferred embodiment, the lipid is a phospholipid having a double hydrocarbon tail.
The hydrophilic polymer R1 can be, in yet another embodiment, selected from the group consisting of polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide, polymethacrylamide, polydimethyl-acrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, copolymers thereof, and polyethyleneoxide-polypropylene oxide.
In one preferred embodiment, the hydrophilic polymer R1 is polyethyleneglycol. In another embodiment, when R1 is polyethylene glycol, R5 is H and R2 is CH3 or C2H5 
In still another embodiment, the amine-containing ligand R4 is a polypeptide. The polypeptide can be, in another embodiment, a recombinant polypeptide. Exemplary and preferred polypeptides include cytokines, such as interferons, interleukins, and growth factors, and enzymes.
In another aspect, the invention includes a composition comprising a conjugate obtainable by reaction with a compound having the general structural formula: 
wherein R1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety; R2 is selected from the group consisting of H, alkyl and aryl; R3 is selected from the group consisting of O(Cxe2x95x90O)R4, S(Cxe2x95x90O)R4, and O(Cxe2x95x90S)R4; R4 comprises a leaving group; and R5 is selected from the group consisting of H, alkyl and aryl; and where orientation of CH2xe2x80x94R3 is selected from the ortho position and the para position. The composition also includes a pharmaceutically-acceptable carrier, such as saline, buffer or the like.
In one embodiment of this aspect, R2 is selected from the group consisting of CH3, C2H5 and C3H8.
In another embodiment, R3 is O(Cxe2x95x90O)R4 and R4 is a hydroxy- or oxy-containing leaving group. The leaving group, in another embodiment, is derived from a compound selected from the group consisting of chloride, para-nitrophenol, ortho-nitrophenol, N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide, N-hydroxyglutarimide, N-hydroxynorbornene-2,3-dicarboxyimide, 1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine, 2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, immidazole, triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol and trichlorophenol.
In one embodiment, the claimed compound is reacted with an amine-containing ligand that displaces R4 to form a conjugate that includes the amine-containing ligand. For example, the amine-containing ligand can be a phospholipid.
In a preferred embodiment, the hydrophilic polymer R1 is polyethyleneglycol, R5 is H and R2 is CH3 or C2H5.
In yet another aspect of this embodiment, the composition containing the conjugate comprises a liposome. The liposome can further comprise an entrapped therapeutic agent.
In another embodiment, the amine-containing ligand comprises a polypeptide.
In yet another aspect, the invention includes a liposome composition comprising liposomes which include a surface coating of hydrophilic polymer chains wherein at least a portion of the hydrophilic polymer chains have the general structure: 
wherein R1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety; R2 is selected from the group consisting of H, alkyl and aryl; R3 is selected from the group consisting of O(Cxe2x95x90O)R4, S(Cxe2x95x90O)R4, and O(Cxe2x95x90S)R4; R4 comprises an amine-containing ligand; and R5 is selected from the group consisting of H, alkyl and aryl; and where orientation of CH2xe2x80x94R3 is selected from the ortho position and the para position. The liposomes have a longer blood circulation lifetime than liposomes having hydrophilic polymer chains joined to the liposome via an aliphatic disulfide linkage.
In one embodiment, the liposome further comprises an entrapped therapeutic agent.
In still another aspect, the invention includes a method for improving the blood circulation lifetime of liposomes having a surface coating of releasable hydrophilic polymer chains. The method includes preparing liposomes that have between about 1% to about 20% of a compound having the general structure: 
wherein R1, R2, R3, and R5 are as described above and R4 comprises an amine-containing lipid.
In a preferred embodiment of this aspect, R5 is H and R2 is selected from the group consisting of CH3, C2H5 and C3H8.
In another embodiment, the amine-containing lipid comprises a phospholipid and R1 is polyethyleneglycol.
In this aspect, the liposomes can further comprise an entrapped therapeutic agent.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.